Abstract
The dual color localization microscopy (2CLM) presented here is based on the principles of spectral precision distance microscopy (SPDM) with conventional autofluorescent proteins under special physical conditions. This technique allows us to measure the spatial distribution of single fluorescently labeled molecules in entire cells with an effective optical resolution comparable to macromolecular dimensions. Here, we describe the application of the 2CLM approach to the simultaneous nanoimaging of cellular structures using two fluorochrome types distinguished by different fluorescence emission wavelengths. The capabilities of 2CLM for studying the spatial organization of the genome in the mammalian cell nucleus are demonstrated for the relative distributions of two chromosomal proteins labeled with autofluorescent GFP and mRFP1 domains. The 2CLM images revealed quantitative information on their spatial relationships down to length-scales of 30 nm.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adenosine Triphosphatases / genetics
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Adenosine Triphosphatases / metabolism
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Cell Line, Tumor
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Cells / ultrastructure*
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Chromatin / metabolism
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Chromatin / ultrastructure*
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Chromatin Assembly and Disassembly / physiology
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Chromosomal Proteins, Non-Histone / genetics
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Chromosomal Proteins, Non-Histone / metabolism
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Computer Simulation
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Green Fluorescent Proteins / genetics
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Green Fluorescent Proteins / metabolism
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Histones / genetics
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Histones / metabolism
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Humans
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Image Processing, Computer-Assisted / methods*
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Luminescent Proteins / genetics
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Luminescent Proteins / metabolism
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Microscopy, Fluorescence / methods*
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Red Fluorescent Protein
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Software
Substances
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Chromatin
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Chromosomal Proteins, Non-Histone
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H2AX protein, human
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Histones
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Luminescent Proteins
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Recombinant Fusion Proteins
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Green Fluorescent Proteins
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Adenosine Triphosphatases
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SMARCA5 protein, human